The potent neuromodulatory roles of NO emerge from both regulation of its synthetic enzyme and its own diverse effects on neural function. Major aspects of the regulation, actions and integrative function of this critical cellular modulator continue to be uncovered. In a simple model system the regulation and modulatory role of NO production are approachable at levels that relate behavior, cell signalling pathways and ion channels. NO synthase (NOS) is found in identified and well characterized neurons of the feeding motor network, an is up-regulated by serotonin (5-HT), itself a physiological arousal factor. NO regulates excitability of the feeding neurons and network arousal state by potentiating a prominent cAMP-gated cation current (INacAMP). NOS activity varies with satiation state, acting like a homeostatic regulator of feeding behavior. This proposal has three specific and integrated aims: 1) exploring the dynamic regulation of NOS by Ca2+, calmodulin, pHi and arginine substrate in single, physiologically defined cells, 2) probing the signalling pathways through which 5-HT affects the activity and expression of NOS in single identified neurons, and 3) elucidating cellular and kinetic mechanisms by which NO regulates neuron excitability via cAMP-gated Na2 current. A major procedural strength is that these studies will take place in single identified and living neurons of well defined functional roles, for which new and potent methods for assessing NOS activity in single neurons are combined with conventional biochemical and pharmacological assays. In elucidating the neural substrates of cellular plasticity in which NO action is embedded, this proposal has fundamental relevance to understanding mechanisms of learning and memory and their disorders, hyperexcitability and depression, and motor and sensory dysfunction. It has applied relevance to understanding physiological consequences of altered 5-HT levels in integrated neural network functions that result in altered mood and affective behavior.